Optical tweezers are an extremely useful
tool in the fields of cellular
and
molecular biology. In the lab of Dr. Bahman Anvari, I use the
optical tweezers
to study intermolecular forces of various receptor-ligand interactions
associated
with the cardiovascular system. Thrombosis is one of the leading causes
of sickness and death and is clinically more important than all of the
hemorrhagic
disorders combined. Thrombi can form in any part of the
cardiovascular
system and appear due to some type of endothelial cell
perturbation.
While the endothelial cells are involved in the initial stage of
thrombus formation,
the main participants in thrombogenesis are the platelets.
Platelets
adhere, alter their shape, and secrete their granular contents in
thrombogenesis.
Numerous receptors on the surface of the platelet interact with various
ligands causing adhesion, platelet aggregation, and a change in
platelet shape.
The glycoprotein (GP) Ib-IX-V complex is the protein on the platelet
surface
that initiates thrombus formation. GP Ib-IX-V mediates the
deposition
of platelets on the subendothelium in a damaged area and initiates
thrombosis
by binding under high shear conditions (as in regions of a damaged
vessel) to a
multimeric protein called von Willebrand factor (VWF) that circulates
in the plasma.
By quantitatively evaluating the VWF/GP Ib-IX-V bond strength using
optical
tweezers, we can gain new insight into the individual molecular
interactions
involved in thrombogenesis and eventually develop new drugs that target
specific
stages in the formation of thrombi.Using our optical
tweezers,
I examine the intermolecular force between GP Ib-IX-V and VWF. As a
model system,
we use transfected Chinese hamster ovary cells that express the
platelet glycoprotein
receptor. In addition, we use polystyrene beads coated with VWF. In our
experiments, we trap the coated bead and let the bead and the cell
adhere for
a certain amount of time. We then use a piezo-electric stage to detach
the cell
from the bead. Using Stokes's Law, we can compute the binding force
between
VWF and GP Ib-IX-V. We use a titanium sapphire laser tuned to a
wavelength of 830
nm to minimize cellular damage. Multimers of VWF that are of average
size do
not normally bind platelets so we use the exogenous modulators
ristocetin and
botrocetin to induce VWF/GP Ib-IX-V adhesion. Our
results are very
interesting because we notice that the detachment forces of the bead
from the cell
occur in quantal units. Other groups, which investigate
different receptor-ligand
interactions using optical tweezers, also frequently observe discrete
force quantities associated with the rupture of bonds. I have also
examined
the bindi color(255,255,255);ng of
unusually large forms of VWF to GP Ib-IX-V and the role
that various
amistyle="top: 0px; height: 1600px;"no acids of the platelet receptor
complex play in
adhesion. In future,
I plan to investigate adhesion of different mutants of GP Ib-IX-V to
VWF
and also determine the significance of loading rate when detaching the
bead from
the cell.